Redox homeostasis is defined as the endogenous ability of the cells in dealing with challenges from various stressors generated by metabolic activities or any exogenous sources. Persistent exposure to the stressor and inadequate ability of the cells to restore the balance leads to oxidative stress formation. Hence, restoration of the redox homeostasis is essential to prevent oxidative stress conditions. For this purpose, the cells increase production of oxidants that acts as signaling species resulting in balance equilibrium in production of pro-inflammatory reaction and pro-oxidant enzymes. The whole process is only aimed to diminish the challenge and restore the homeostasis (Ursini et al., 2016).

In the eye, ROS are not only generated endogenously due to robust metabolic activities. Intense exposure to light, and high atmospheric oxygen exposure also contributed to generation of ROS in the eye (Beatty et al., 2000; Tokarz et al., 2013). Neutralization of this abundantly generated ROS in the eye is crucial for cellular homeostasis. At a low and moderate level, the ROS acts a signaling molecule that sustain cellular proliferation and differentiation as well as activate stress-responsive survival pathways by increasing the expression of pro-oxidant related genes. Persistent exposure to ROS would implicated to various eye diseases such as cataract (Ho et al., 2010), glaucoma (Izzotti et al., 2006), and AMD (Beatty et al., 2000). GSH, NADH, and NADPH are examples of the cellular redox presented in the eye mainly generated from mitochondria-related pathways and their level are reduced with aging (Bradshaw, 2019; Pan et al., 2021).

Thymoquinone has antioxidant capacity that has been found to be able to maintain redox homeostasis in the cells. Thymoquinone has been shown to decrease the level of GSH and increase the level of NADH and NAPDH (Sankaranarayanan and Pari, 2011). These molecules serve important roles in biological oxidation-reduction processes, as a cofactor for many enzymes, and a detoxifying and free radical scavenging agent, hence retain the cell homeostasis. GSH is responsible in maintaining the sulfhydryl (–SH) group of enzymes in its reduced state. The sulfhydryl residues of GSH molecules are easily oxidized to form GSH disulfide (GSSG), which is then reduced back to GSH by the reaction with GSH reductase (Aoyama and Nakaki, 2015). Since RNS and ROS may interact with cysteine thiol group to form electrophile containing –SH, GSH is responsible to attack these molecules and retain it at less reactive state (Armutcu et al., 2018). Decrease in GSH level is accompanied with increase in GSSH is observed in oxidative stress related diseases which include AD and PD (Calabrese et al., 2006; Mischley et al., 2016). The redox capacity of cells is determined by the ratio of GSH/GSSG. In an in vivo study, decreased level of GSH was observed in the brain tissue treated with sodium nitrite and it was restored with thymoquinone treatment. In addition, thymoquinone treatment also shown to decrease the level of TNF-α and IL1β and increase level of IL-10 in a concentration-dependent manner (Hamdan et al., 2019). Similar reaction was observed in the model of brain ischemia (Al-Majed et al., 2006). Mass spectrometry analysis suggest that thymoquinone react with GSH by rapid binding of the thymoquinone to the cysteine residues of the GSH molecules in the 3-position of benzoquinone ring, hence prevent GSH oxidation to GSSG (Khalife and Lupidi, 2007).

NADH is the reduced form of NAD+ plays important role in mitochondrial metabolism. The NAD+/NADH ratio is a known regulator of various biological processes in the cells including glycolysis, energy metabolism, mitochondrial function, and gene expression (Xiao et al., 2018). The NADH is essential for ATP production and oxidative phosphorylation in the electron transport chain system. Decrease in the NAD+/NADH ratio, reduced cellular NAD + level, and increased NADH have been observed during aging (Braidy et al., 2011). NADPH is responsible for maintaining cells’ redox state and biosynthesis of fatty acids, cholesterol, and deoxynucleotides. NADPH decreased in aging as a result of decreased of its immediate precursor, NADP+. Therapies that increase NAD + levels and NADP + could greatly benefit subjects with aging-related disorders. Study in BV2 microglial cells showed that thymoquinone was able to reduce LPS-induce ROS by increasing the level of NAD+/NADH ratio. This reaction is followed by the attenuation of cellular ROS level (Velagapudi et al., 2017a). In addition, thymoquinone was also showed to play a major role in hyperglycemia model which is important in DR pathogenesis. A significant drop of 45 and 30% of NADH and NADPH was observed with 5 μm of thymoquinone incubation (Gray et al., 2016). The mechanism on how thymoquinone regulate the NAD+, NADH, NADP+, and NADPH is still not clear. Some research suggest it happens through inhibition of p47PHOX, a cytosolic subunit of the NADPH oxidase, phosphorylation on Ser-304 and Ser-328 (Boudiaf et al., 2016).

Oxidative stress has long been linked to aging and neurodegenerative disorders, including those affecting the eyes (Jadeja and Martin, 2021). An imbalance in the accumulation of harmful substances in cells and the cell’s normal ability to avoid oxidative damage by using its anti-oxidative properties is referred to as oxidative stress (Beatty et al., 2000; Burton and Jauniaux, 2011; Zafrilla et al., 2013). Under normal homeostasis, the antioxidant would neutralize the oxidative stress level to ensure cell survival. Persistent stress, on the other hand, will disrupt this protective mechanism, causing cells to undergo apoptosis, necrosis, or autophagic cell death. Oxidative stress is accompanied by the increase in ROS in the cells. The major types of ROS are superoxide anion, hydrogen peroxide, hydroxyl radical, peroxy radical, and the second messenger nitric oxide (Burton and Jauniaux, 2011). A major source of ROS production in cells is the mitochondrial respiratory chain. Oxidative stress accumulation in cells is detrimental because it causes cellular damages to proteins, lipids and nucleic acids (Cui et al., 2012). Excessive accumulation of ROS can trigger downstream signaling pathways such as NF-κB, ERK1/2, p38 MAPK, and autophagy-related signaling (Zhang et al., 2016).

Due to a variety of factors, including high oxygen intake, excessive light exposure, and the presence of polyunsaturated fatty acids, the retina is extremely susceptible to oxidative stress harm. Furthermore, since the outer segments of photoreceptors are constantly shed, the RPE’s phagocytic role adds to the oxidative burden (Beatty et al., 2000; Tokarz et al., 2013). The accumulation of oxidative stress disrupts cell homeostasis and triggers a defensive cell response in photoreceptors and retinal ganglion cells (Nita and Grzybowski, 2016). These two cell types are high-metabolizing neurons that are constantly exposed to oxidative stress insults (Beatty et al., 2000).

Thymoquinone is known to reduce in vitro and in vivo oxidative stress levels. Various studies showed the ability of thymoquinone to reduce oxidative stressors such as TNFα, lipopolysaccharides, IL-1β and Il-1α levels (Wang D. et al., 2015; Cobourne-Duval et al., 2018). In the case of neurodegenerations, Sedaghat et al. (2014) has shown that thymoquinone was able to ameliorate the oxidative stress marker [malondialdehyde (MDA) and nitrile] levels in unilateral intrastriatal 6-hydroxydopamine (6-OHDA)-lesioned rats as a model of PD (Sedaghat et al., 2014). In another study, thymoquinone significantly decreased MDA, nitrile oxide and TNF-α level in an AD model (Abulfadl et al., 2018b). A similar observation was made in LPS-induced oxidative stress in BV2 microglialcell, where 10um of thymoquinone was able to reduce the inflammatory cytokines expressions through qPCR and ELISA (Cobourne-Duval et al., 2018). In addition, thymoquinone treatment (2.5, 5, and 10 mM) was shown to reduce the levels of TNF-a, IL-6, and IL-1b in BV2 cells challenged by LPS at both mRNA and protein levels (Velagapudi et al., 2017a). Thymoquinone (3, 6, 12 μM) was found to exert its effect by reducing the level of nitrite oxide and PGE2 production indicated in BV2 cells (Wang Y. et al., 2015). On the contrary however, Elibol et al. (2019) showed that in the case of AD thymoquinone did not change the level of TNF-α, IL-1α, and IL-1β groups with pretreatment of thymoquinone (Elibol et al., 2019).

In addition to reducing oxidative stressors, thymoquinone has been shown to increase inherent intracellular antioxidants such as SOD, catalase and glutathione. In an in vivo study of arsenic-induced neurotoxicity, 10 mg/kg of thymoquinone suppressed levels of oxidative stressors (NO and TNF-α) and increasing antioxidants (glutathione, glutathione peroxidase, SOD, and catalase) in the rat’s nervous system (Kassab and El-Hennamy, 2017). The same effect was seen in lead (Mabrouk, 2017) and paraquat toxicity (Zeinvand-Lorestani et al., 2018) in rat’s liver. Co-treatment with 5 mg/kg/day of thymoquinone for 5 weeks resulted in increased in reduced glutathione levels, but also SOD, glutathione peroxidase, catalase and glutathione reductase activities (Mabrouk, 2017). Meanwhile, pretreatment with 10 mg/kg thymoquinone was shown to restore SOD activity in paraquat-induced toxicity (Zeinvand-Lorestani et al., 2018). These studies show the ability of thymoquinone in ameliorating the level of antioxidant in hepatotoxicity injury.

The effect of thymoquinone on different cell types may vary and possibly are cell specific. However, it is possible that thymoquinone provide protective effects on the retina by reducing oxidative stress and increasing antioxidant properties. Therefore, it is worthwhile to examine the effects of thymoquinone on ocular neurodegenerative models.

Inflammatory response occurs as a result of modulation of oxidative stress involving a series of cellular events that allow the body to defense itself from intruders or further damage (Hussain et al., 2016). Accumulation and prolong exposure to oxidative stress triggers exaggerated inflammatory response of cells termed as chronic inflammation. Studies showed that chronic inflammation does play an important role in age-related disease (Sharif et al., 2019). Inflammation is considered as the primary immune system reaction to eliminate pathogens or other stimuli in order to restore the cells to normal state (Chen et al., 2017).

In the eye, there five neuronal cells present namely photoreceptors, bipolar cells, ganglion cells, horizontal cells, and amacrine cells. These cells are responsible in providing vision. With aging, these neuronal cells undergo various physiological and morphological changes due to various endogenous and exogenous factors including oxidative stress and inflammation, which leads to impaired vision. Recently, inflammation has become a concern for AMD, DR and macular edema as a secondary to DR (Das, 2016; Tan et al., 2020). The importance of inflammation in these diseases has been discussed (Ascaso et al., 2014; Kauppinen et al., 2016; Forrester et al., 2020). Apart from anti-VEGF treatment, anti-inflammatory agents such as corticosteroids has been used to delay the progression of these disease (Wang et al., 2011). Macrophages and giant cells have been reported to localize near drusen, at the breakdown of Bruch’s membrane, and in the CNV membrane of AMD. Furthermore, increase in inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and IL-1, was also found in the RPE, thereby inducing additional inflammatory cellular infiltration and accelerates angiogenesis and CNV formation in AMD (Wang et al., 2011). VEGF is a growth factor responsible in promoting angiogenesis. However in DR and AMD, the presence of VEGF cause uncontrolled growth of blood vessels and promote disease progression. Increase of VEGF and its involvement in ocular disease is well studied (Homayouni, 2009; Ahuja et al., 2019).

Microglia activation is considered first line of defense against inflammation followed by release of anti-inflammatory mediators and resolution of the inflammatory response. It is well known that microglia activation is an important in pathogenesis of AD and PD (Qian and Flood, 2008; Mandrekar-Colucci and Landreth, 2010). Analysis on microglia cells exposed to thymoquinone demonstrated a significant anti-inflammatory property of thymoquinone observed through attenuation of IL-6, CCL12/MCP-5, and CCL2/MCP-1 protein and mRNA expression (Taka et al., 2015). In another study, thymoquinone showed anti-inflammatory properties by inhibition of nitric oxide (NO), PGE2, TNF-α, and IL-1β production in BV2 microglial cells (Wang Y. et al., 2015). Besides, thymoquinone anti-inflammatory activity in AD has been observed through alleviation of TLR signaling pathway, a signaling pathway responsible for innate response. Different thymoquinone dosage ranging from 10 mg/kg/day to 40 mg/kg/day was able to reduce TLR-2, TLR-4, MyD88, TRIF and IRF-3, which are related to TLR signaling (Abulfadl et al., 2018a). In conclusion, these findings suggest a possible anti-inflammatory effect of thymoquinone through attenuation of various inflammatory related cytokines, which could provide therapeutic value in neurodegenerative diseases and ocular disease.

Accumulation of oxidative stress are reported to cause peroxidation of polyunsaturated fatty acids in the cell membrane of the brain resulting in the formation of toxic metabolites such as MDA and 4HNE, as well as advanced glycation end products (AGEs). Long-lived proteins and protein deposits in human and animal tissues, including the retina, have been shown to contain these byproduct metabolites. The AGEs is a byproduct of Maillard reaction through interaction of aldehyde or carbonyl sugar with amino group of proteins, lipids or nucleic acids (Goldin et al., 2006; Luevano-Contreras and Chapman-Novakofski, 2010). In the eye specifically, AGEs are associated with age-related macular degeneration (AMD), cataract formation, diabetic retinopathy and glaucoma (Ishibashi et al., 1998; Howes et al., 2004).

Advanced glycation end products accumulation can affect the eye through two mechanisms, either by damage to the extracellular matrix layer or interactions with receptor proteins. AGEs accumulate with age (Araki et al., 1992; Schleicher et al., 1997). Accumulation of AGEs in the eye would leads to abnormal crosslinking of extracellular matrix proteins, increase vascular stiffness, leading to changes in vascular structure and function. As a result, damage and leakage in the blood vessels and hyperproliferation can occur in diabetic retinopathy and AMD (Vlassara et al., 1994). AGEs formation is enhanced in hyperglycemic conditions in diabetic patients and reflects the severity of diabetic complications (Ono et al., 1998). Localization of AGEs in retinal blood vessels has been observed in patients with type 2 diabetes (Stitt et al., 1997). AGEs-induce oxidative stress cause consequent apoptosis of retinal pericytes (Bergers and Song, 2005). Pericytes play an important role in the maintenance of microvascular homeostasis. Loss of pericytes leads to angiogenesis, thrombogenesis, and endothelial cell injury, thus leading to diabetic retinopathy development (Eilken et al., 2017). In addition, AGEs interact with RAGE, resulting in increased intracellular production of free radicals and oxidative stress, which is then phosphorylates by MAP kinase and activates and increases expression of NF-κβ controlled genes causing vasoconstriction, angiogenesis, increased adhesion molecule expression, and inducing a procoagulant state associated with AMD and diabetic retinopathy (Tong and Yao, 2006; Sun et al., 2017).

There has been growing evidence suggesting that thymoquinone may be attributed to reducing the levels of AGEs (MDA, 4HNE) in cells. Administration of thymoquinone (10 mg/kg) in AD models has shown to lower the MDA level (Zaher et al., 2019). Besides, the protective effect of thymoquinone through reduction of 4HNE was observed in the model of renal (Dera et al., 2020a) and pulmonary diseases (Dera et al., 2020b). Reduction of MDA was also accompanied by increased levels of SOD, catalase and reduced glutathione (GSH). The findings were supported by an in vitro study Alhebshi et al. (2013), which showed that thymoquinone administration restored the redox balance in cultured rat hippocampal and cortical neurons displaying Aβ aggregation, as seen in AD (Alhebshi et al., 2013). Several studies also showed the same results in various condition such as in the liver and kidney of the rat model (Hosseinian et al., 2017), spinal cord injury model (Chen et al., 2018), diabetic model (Desai et al., 2015), renal induced toxicity (Sener et al., 2016), and ischemic brain model (Xiao et al., 2016).

Blocking of the Maillard reaction has been shown to be beneficial in preventing AGEs formation. Thymoquinone acts on AGEs by blocking the attachment of sugar to proteins, thereby attenuating glycosylation and oxidative stress while preventing protein crosslinking in a dose dependent manner (Losso et al., 2011; Anwar et al., 2014). Several studies have been shown to reduce glycation in sugar-induced AGEs model following administration of thymoquinone (Losso et al., 2011; Mahmood et al., 2013; Zafar et al., 2013; Benvidi et al., 2018; Pandey et al., 2018). Mahmood et al. (2013) and Zafar et al. (2013) observed the effect of glucose in inducing AGEs in model of diabetic. Both studies showed thymoquinone’s ability in reducing AGEs formation measured through thiobarbituric assay and AGEs fluorescence read at 480 nm (Mahmood et al., 2013; Zafar et al., 2013). In cases of fructose and BSA glycation, Pandey et al. (2018) shows thymoquinone reduced browning effect, indication of glycation process during AGEs formation (Pandey et al., 2018). The group also looked into carbonyl content and glycation aggregation index, which is a part of late stage of AGEs formation process. Data showed thymoquinone can reduce glycation aggregation index.

In support of those study, Losso et al. (2011) assessed the potential inhibitory effect of thymoquinone against AGEs from serum samples of diabetic patients through the use of hemoglobin-δ-gluconolactone, human serum albumin-glucose and the N-acetyl-glycyl-lysine methyl ester-ribose assay. Their results suggested thymoquinone at a dose as low as 10mM was able to suppress 78% of AGEs in blood plasma (Losso et al., 2011). The effect of thymoquinone on AGEs was also linked to the binding of thymoquinone to arginine-409 of thymoquinone as observed in BSA-induced AGEs (Benvidi et al., 2018). In addition, reduction of AGEs formation was also postulated to be due to the ability of thymoquinone in preventing the inactivation of SOD, an enzyme responsible as a defense mechanism in prevention of glycation (Anwar et al., 2014; Benvidi et al., 2018). Incubation of SOD with glycating agent, glucose, methyglyoxyl or both, has been shown to increase structural changes by SOD, thus alter it defense mechanism. However, thymoquinone was shown to reverse the effect by reducing the amount of SOD structural changes, attenuate AGEs formation as well as reduced protein crosslinking (Anwar et al., 2014). These data show the important of thymoquinone as an anti-glycating agent that may be useful to tackle AGEs formation in the ocular disorder.

Apoptosis is defined as a programmed cell death intend to remove unwanted or damaged beyond repairs cells in the body. Shrinkage of the cell, fragmentation into membrane-bound apoptotic bodies and rapid phagocytosis by neighboring cells are the common characteristic of apoptosis (Nickells and Zack, 1996). Under normal circumstances, apoptosis is important to prevent accumulation of unwanted cells and making a way for new young cells to grow. Dysregulation of apoptosis can cause health problems. In cancer for instance, apoptosis process is absent followed with uncontrolled growth of new cells. In addition, excessive apoptosis also can create degeneration conditions in the body as seen in AD and PD, where the brain lose some part that is not supposed to die.

In the eye, apoptosis is considered a mechanism of cell death in ocular diseases including glaucoma, retinitis pigmentosa, cataract, AMD and diabetic retinopathy. Apoptosis has been found precedes these disease progression (Charakidas et al., 2005; Feenstra et al., 2013). DNA fragmentation analysis using terminal digoxigenin-labeled dUTP nick end labeling (TUNEL) in cataract patient revealed that apoptosis in cataract patient is higher compared to non-cataract patient. 92.6% of the samples tested shown average of 21.7 apoptotic cells per mm2, or 0.0047% of overall cell population and only three apoptotic epithelial cells per mm2 was detected from the non-cataract samples (Charakidas et al., 2005). Analysis of apoptosis modulator, Bcl-2 and Bax would allow determination of the susceptibility of a cell to apoptosis. Increase in the expression of both these proteins during disease progression was observed in glaucoma (Zalewska et al., 2004), retinitis pigmentosa (Comitato et al., 2014), cataract (Osnes-Ringen et al., 2016), AMD (Dunaief et al., 2002), and diabetic retinopathy (Khalfaoui et al., 2010). These findings suggest the important of apoptosis in the ocular disease development and study on possible modulation of these important process may leads to a crucial opportunity to deliver treatment sooner and hence preserve vision.

Apart from the earlier mentioned protective capability of thymoquinone, it also serves a dual function in apoptosis. In cancer cells, thymoquinone has been shown to promote apoptosis. In bladder cancer, thymoquinone was showed to induce apoptosis by enhancing the expression of the anti-apoptotic protein Bcl-2, blocking the release of cytochrome c and enhance the translocation of Bax from the cytoplasm to mitochondria (Zhang et al., 2018). Similar reaction was observed in breast cancer. Proapoptotic capacity of thymoquinone was observed through inhibition of anti-apoptotic genes, such as XIAP, survivin, Bcl-xL and Bcl-2, and increase in DNA fragmentation (Woo et al., 2013). Further study showed that pro-apoptotic activity of thymoquinone is followed with reduction in telomerase activity, an enzyme responsible for cellular resistance to apoptosis (Gurung et al., 2010). Thus, inhibition of telomerase by thymoquinone may induce apoptosis which is essential to prevent cancer.

On contrary, thymoquinone has been found to regulate apoptosis by reducing DNA fragmentation, downregulating of Bax and upregulating the Bcl-2 expression as well as decreasing downstream caspases 8, 9, and 3 in model of hepatic ischemia reperfusion injury, hence blocking apoptosis (Abd El-Ghany et al., 2009). In an in vitro model of AD, thymoquinone was shown to prevent apoptosis by blocking αβ-induced neurotoxicity (Alhibshi et al., 2019). Treatment with αβ_1–42 for 48 h showed a significant decreased in cell viability to 63.5% as compared to the control group and cotreatment with 100 nM of thymoquinone restored the cell viability to 90%. The restoration of cell viability by thymoquinone was accompanied with significant reduction in ROS level to 2.6-fold as compared to sixfold in αβ_1–42 only treatment. Ismail et al. (2013), also observed similar results with additional preserved intact cell bodies, extensive neurite networks, a loss of condensed chromatin and less free radical generation in primary cultured cerebellar granule neurons than those exposed to Aβ1–40 alone (Ismail et al., 2013). From these data, it is shown the selective capability of thymoquinone in inducing or inhibiting apoptosis in cells. This raises another interesting topic in studying the mechanism of thymoquinone in cells and applied it to ocular research.

Apoptosis is closely related to mitochondrial dysfunction. Mitochondrial dysfunction is described as a decrease in electron transport chain efficiency and a decrease in the synthesis of high-energy molecules like adenosine-5′-triphosphate (ATP). Mitochondrial dysfunction is a common feature in aging, and it also occurs in chronic diseases such as AD, muscular dystrophy, diabetes and cancer. It is important to note that ROS is primarily produced in the mitochondria, where they inhibit the mitochondrial electron transport chain’s complex I and reduce membrane integrity, resulting in higher ROS production (He et al., 2008). Mitochondrial-related mutation in the eye was first observed in Leber hereditary optic neuropathy (LHON). Mitochondrial DNA mutation in LHON affects the complex I of the electron transport chain, altering ATP synthesis. The resulting retinal ganglion cell apoptosis and optic nerve injury subsequently leads to blindness (Ghelli et al., 2003).

The mitochondria in retinal ganglion cells generate a large amount of energy required for nerve function in the unmyelinated ganglion cell axons. Glaucoma, like LHON, is an optic nerve disease characterized by accelerated apoptosis of retinal ganglion cells and their axons. It has been hypothesized that during the onset of glaucoma, an imbalance in the retinal ganglion cell axon energy requirement is caused by a change in blood flow dynamics in the optic nerve head, rendering the ganglion cells vulnerable to additional insults such as light entering the eye, which can further affect ganglion cell axon mitochondrial activity, and molecules released by astrocytes. If the mitochondria are unable to maintain normal function in these conditions, ganglion cell death can occur. Loss of retinal ganglion cells in glaucoma mouse models has been documented and this loss is due to the reduction in quantity of mitochondria and impairment of the mitochondrial function (Kim et al., 2015; Takihara et al., 2015).

More recently, the role of mitochondrial damage was investigated in diabetic retinopathy and AMD. An important cause of these conditions is an excess of pro-oxidants induced by the overproduction of ROS by the mitochondria throughout the retina. Increased retinal mitochondrial damage and disrupted mitochondrial membrane were observed in diabetic mouse models, together with elevated levels of ROS (Kanwar et al., 2007). In addition, in a diabetic rat model, the retinal mitochondria were shown to be leaky with diabetes, hence cytochrome c began to accumulate in the cytosol of retinal cells, observed in vitro rat models (Kowluru and Abbas, 2003). The RPE contains a high density of mitochondria to produce sufficient ATP to perform all its physiological processes. As a result, age-related mitochondrial dysfunction will lead to an increase in oxidative stress in the RPE, resulting in AMD. Wang et al. (2008) showed that mitochondrial damage was increased with age in the RPE of mice and rats (Wang et al., 2008). They concluded that the increase in mitochondrial damage in aging was due to a decrease in the cell’s DNA repair capability. In the case of AMD, Ferrington et al. (2017) and Golestaneh et al. (2017) investigated the mitochondrial function in RPE of AMD and healthy donor RPE. Their results showed reduction in mitochondrial function in RPE of AMD patients as compared to healthy donors (Ferrington et al., 2017; Golestaneh et al., 2017). It is also important to note, mitochondrial dysfunction can result in further increase in oxidative stress and thus leads to RPE functional decline and apoptosis. Taking these data together, maintaining the mitochondrial function and integrity is important to reduce the damage to the retinal ganglion cell and RPE cells in order to prevent blindness.

Like in the retina, the energy requirement in the brain is high to maintain its physiological function, thus mitochondrial dysfunction may pose a threat and lead to neurodegeneration as seen in AD and PD (Picone et al., 2014). The effect of thymoquinone on arsenic induced hippocampal toxicity and mitochondrial dysfunction in Wistar rats was studied (Firdaus et al., 2018). Arsenic is a compound found in pollutants and was found to induce oxidative stress as well as interruption in mitochondrial functions. Exposure to arsenic caused significant increase in intracellular ROS generation, mitochondrial dysfunction and apoptotic events. Administration of thymoquinone (2.5 and 5 mg/kg) was shown to inhibit the mitochondrial dysfunction by reducing the mitochondrial membrane potential in the rats hippocampal. Administration of thymoquinone concentration of 30 mg/kg for 10 consecutive days was shown to prevent mitochondrial dysfunction in liver of warm ischemia-reperfusion (I/R) injury rat model (Bouhlel et al., 2017, 2018). Thymoquinone was shown to also lessen the release of cytochrome C, an activator for caspase 9 and caspase 3 apoptotic pathways and thus preserve the mitochondrial function and prevent cell apoptosis. The prevention of liver mitochondrial damage could also be explained by the antioxidant and free radical scavenging activities of thymoquinone which in turn stabilized mitochondrial membrane permeability. Khan et al. (2012) showed the protective effect of thymoquinone against mitochondrial dysfunction in an in vitro model of AD using differentiated PC 12 cell line (Khan et al., 2012). In their study, thymoquinone pretreatment (1, 2, 4 μm) significantly counteracted the decreased in mitochondrial membrane potential. This showed an important mechanism of thymoquinone in combating mitochondrial dysfunction in cells. In addition, administration of 10 mg/kg of thymoquinone was shown to increase ATP synthesis in the renal cortex of kidney disease models (Dera et al., 2020a). These data together show a promising use of thymoquinone in maintaining retinal mitochondrial integrity and functions to prevent ocular neurodegeneration.

The cells undergo cell-to-cell communication through signaling pathways. Various signaling pathways have been identified and associated with different diseases in the eye. Among all, NF-κβB is widely studied due to its important role in regulating key cellular processes such as inflammation, apoptosis, stress response, wound healing, and angiogenesis (Morgan and Liu, 2011). NF-κβ is a transcription factor present ubiquitously in the cell cytoplasm as a dimer bound to an inhibitory complex, Iκβ. NF- κβ molecule activation is triggered by a variety of stimuli including growth factors, cytokines, bacterial lipopolysaccharides, UV, and oxidative stress. Activation and translocation of NF-κβ molecules involved a series of phosphorylation and ubiquitination processes leading to gene transcription through binding of the NF-κβ dimer to the κβ sites of DNA sequences. Binding of NF-κβ dimer to the κβ sites of DNA sequences NF-κβ will induce gene transcription of specific proteins including various cytokines (Lingappan, 2018). NF-κβ is thought to be cell specific and it can even exert a dual function, for cell survival and induce cell death.

In the eye, the NF-κβ signaling is activated by retinal degeneration (Wu et al., 2002; Zeng et al., 2008). Zeng et al. (2008) studied the alteration of NF-κβ activity during retinal degeneration in rd mice (Zeng et al., 2008). Increase of phosphorylated NF-κB P65 proteins and augmented DNA binding activity were observed in rd mice with retinal degeneration. Wu et al. (2002) also demonstrated intense light exposure activates NF-κB in the photoreceptor cells of rd mouse model followed with increase in NF-κβ DNA–binding activity, and increased in expression of mRNA of Iκβα, a target gene of NF-κβ (Wu et al., 2002). Since NF-κβ activation resulted in the production of proinflammatory cytokines such as TNF-a and IL-1, which mediate inflammatory and immune responses, these findings may indicate that altering NF-κβ activity promotes photoreceptor apoptosis in the rd retina by initiating and perpetuating chronic inflammation. This hypothesis is supported by an in vitro study, where 60–70% of the 661W photoreceptor cells were found to undergo apoptosis with light exposure and level of P65 proteins were downregulated (Yang et al., 2007). Pretreatment with minocycline or sulforaphane countered the downregulation of P65 protein and as a result, photoreceptor apoptosis was inhibited. These data highlight NF-κβ as an extremely attractive target for therapeutic intervention in retinal degeneration.

Thymoquinone was shown to reduce activation of NF-κβ in cells. In one of the studies, BV2 microglial cells were stimulated with LPS after thymoquinone incubation. LPS-induced NF-κβ activation was studied by measuring the secretion of TNFα and other NF- κβ related inflammatory cytokines such as IL-6 and IL-1β (Nagi et al., 1999; Wang Y. et al., 2015; Velagapudi et al., 2017a). The levels of these NF- κβ related cytokines were significantly decreased. In addition, exposure of LPS on rat basophil cell lines showed a marked increase in TNFα production, and thymoquinone was able to reverse this effect at a concentration as low as 10 μM concentration (El Gazzar et al., 2007). Similar findings were noted on studies of thymoquinone on NF-κβ signaling and TNFα cytokines release in the rat’s liver (Sayed and Morcos, 2007).

Thymoquinone (10, 15, 20, 25 μmol/L) suppressed NF-κβ activation in dose dependent manner, specifically by suppression direct binding of nuclear p65 and recombinant p65 to the DNA in 0.1 nmol/L TNFα and LPS stimulated cells. To confirm the finding, cells were transfected with p65 plasmid containing cysteine residue 38 mutated to serine and the binding inhibition by thymoquinone were flattened. Their data showed that thymoquinone suppressed NF-κβ activation correlated with sequential inhibition of the activation of IKK, IKβα phosphorylation, IKβα degradation, p65 phosphorylation, p65 nuclear translocation, and the NF-κβ-dependent reporter gene expression. Expression of NF-κβ regulated antiapoptotic genes (IAP1, IAP2, XIAP Bcl-2, Bcl-xL, and survivin), proliferative (cyclin D1, cyclooxygenase-2, and c-Myc), and angiogenic (matrix metalloproteinase-9 and vascular endothelial growth factor) gene products were also downregulated with thymoquinone treatment (Sethi et al., 2008). Similar findings were reported by Velagapudi et al. (2017b).

Wang Y. et al. (2015) and Velagapudi et al. (2017b) suggested that alteration of NF- κβ by thymoquinone may also be mediated through inhibition of Iκβα phosphorylation or P65 phosphorylation (Wang Y. et al., 2015; Velagapudi et al., 2017b). Thymoquinone has also been reported to exert inhibitory effects on AGE-induced NF-κB-activation in human proximal tubular epithelial cells (pTECs) stimulated with advanced glycation end products (AGEs) (Sayed and Morcos, 2007). All these findings showed the mechanism of thymoquinone in reducing the pro-inflammatory NF-κβ signaling pathways, which can be a reflection of its potential neuroprotective effects in ocular degeneration.

Apart from NF-κβ signaling, thymoquinone also affects – Nrf2 signaling, a master regulator of oxidative stress. Both NF-kB and Nrf2 signaling pathways are inter-related. Increase in NF-κB activation is followed by a decline in Nrf2 signaling pathway, thus further increasing inflammation response. Nrf2 is a transcription factor that is involved in the cellular defense against oxidative stress by controlling the expression of antioxidant-related gene expression resulting in anti-inflammatory response in the cells (Vomund et al., 2017). Nrf2 is thought to play a pivotal role in inflammation because its protein products are responsible for detoxification and elimination of oxidative stress in the cells (Nguyen et al., 2009). Mounting evidence suggests that aging induces a decline in the antioxidant capacity via a reduction in Nrf2 signaling.

The importance of Nrf2 signaling in the pathogenesis of AMD was highlighted when Nrf2-deficient mice developed features similar to human AMD. These mice exhibit pathological characteristics of human AMD including progressive RPE and Bruch’s membrane degeneration, drusen deposits and lipofuscin accumulation, and decreased electroretinography responses (Zhao et al., 2011).

In the RPE, under oxidative stress conditions, the Nrf2 signaling is impaired. Nrf2 transcription factors are lowered, and hence are not able to translocate to the nucleus to induce an antioxidant response. This renders the RPE and photoreceptors vulnerable to damage. Catanzaro et al. (2020) studied the role of Nrf2 on wild type (WT) and Nrf2-silenced (siNrf2)- ARPE-19 cells exposed to various AMD-related noxae (H2O2, 4-HNE, MG132 + Bafilomycin) (Catanzaro et al., 2020). The study showed that in Nrf2 knockout group, cells present a higher susceptibility to oxidative insults as compared to WT groups, suggesting that the Nrf2 induction represents an efficient defensive strategy to prevent the stress-induced damage. Increasing Nrf2-regulated glutathione levels and NAD(P)H attenuated ultraviolet light-induced RPE damage by Gao and Talalay (2004). Carotenoids zeaxanthin and lutein were shown to preserve photoreceptors against light damage by mitigating oxidative stress through Nrf2 signaling (Frede et al., 2017; Yu et al., 2018).

Thymoquinone has been shown to increase activation of Nrf2 signaling. Anti-inflammatory effects of thymoquinone in BV2 microglial cells occurred via the Nrf2 signaling pathway. Activation of Nrf2 signaling by thymoquinone was found to be accompanied by increased nuclear localization, DNA binding and transcriptional activity of Nrf2, as well as increasing protein levels of HO-1 and NQO1. Suppression of Nrf2 activity through siRNA or with the use of trigonelline resulted in the loss of this effect (Velagapudi et al., 2017b). The same results were also observed in models of lung fibrosis (Ahmad et al., 2020), kidney injury (Al Fayi et al., 2020), lung injury (Mao et al., 2020), and allergy response (Dera et al., 2020c). Taken together, these data demonstrated the anti-inflammatory effect of thymoquinone through Nrf2 signaling.

Proteostasis is regulated by a complex network of cellular mechanisms that monitors the concentration, folding, cellular localization, and interactions of proteins from their synthesis through their cellular degradation. Decline in proteostasis is one of the hallmarks of aging and accumulation of damaged protein leads to age related diseases such AD, PD, AMD, glaucoma and cataract. Proteosomes complex is a non-lysosomal ATP-dependent degradation system responsible in removal of damaged, oxidized and misfolded proteins in cells through a process called autophagy (Saez and Vilchez, 2014).

In the eye, autophagy occurs in the lens, cornea, photoreceptors, and RPE cells. Autophagy plays a role in lens fiber cells and corneal maturation (Costello et al., 2013). And maintains the inner segment turnover and survival of the photoreceptor. Within RPE cells, autophagy is involved in aspects of development, cell survival in response to stress, melanin degradation, as well as the degradation of toxic cellular components or damaged organelles. The importance of autophagy in these tissue has been discussed (Frost et al., 2014).

Damaged protein degradation is crucial in cells before the accumulated proteins aggregated. The 20S proteasome is the central core of this system and acts as a part of the intracellular antioxidant defense system (Bonfili et al., 2008). It is estimated that more than 20% of proteins in mammalian cells are degraded by the 20S proteasomes (Baugh et al., 2009). The 20S proteasomes recognized damage proteins through specific degradation signals such as multi-ubiquitin chain tag (Sahu et al., 2021). For a protein to be tagged for degradation, a series of reaction was carried out by E1 (ubiquitin-activating), E2 (ubiquitin-conjugating), and E3 (ubiquitin-ligating) enzymes to create a ubiquitin tag that consist of at least 4 ubiquitin molecules. The chain of ubiquitin directs the damaged proteins to the 20S proteasomes where proteolytic degradation occurs (Ravid and Hochstrasser, 2008; Saez and Vilchez, 2014).

Thymoquinone has been demonstrated to induces selective and time-dependent proteasome complex modulation. However, the mechanism of thymoquinone has only been observed in cancer cells (Cecarini et al., 2010). In glioblastoma cells, thymoquinone has been shown to induce proteosome complex inhibition. This inhibition is due to compromised 20S proteosomes complex functionality and not to downregulation of its synthesis as observed through western blotting and 20S proteosomes activity analysis. In addition, E3 ubiquitin ligase was also downregulated with thymoquinone treatment in acute leukemia model (Alghamdi et al., 2020). Based on this evidence in cancer subject and with the importance of autophagy in the eye, study on thymoquinone in proteosome might provide a novel mechanism of thymoquinone in ocular disease application.